In scanning microscopy, a specimen is illuminated by a light beam in order to observe the reflected light or emitted fluorescent light emanating from the specimen. The focus of an illumination light beam is moved in an object plane using a controllable beam deflection device, generally by tilting two mirrors; the deflection axes usually being perpendicular to one another so that one mirror deflects in the x direction and the other in the y direction. Galvanometer positioning elements are used, for example, for tilting the mirrors. The luminous flux coming from the object is measured as a function of the position of the scanning beam. The positioning elements are typically equipped with sensors in order to determine the active mirror position.
In confocal scanning microscopy, in particular, a specimen is scanned in three dimensions by the focus of a light beam. A confocal scanning microscope generally encompasses a light source, a focusing lens system for focusing the light from the source onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device used for beam control, a microscope lens system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via the beam splitter. The fluorescent or reflected light coming from the specimen travels back via the beam deflection device to the beam splitter, passing through the same, to then be focused onto the detection pinhole, behind which the detectors are located. Detected light not originating directly from the focus region in the specimen travels a different light path and does not pass through the detection pinhole, so that the only information obtained from the focus region is that which leads to the creation of a three-dimensional image by sequential scanning of the specimen. For the most part, a three-dimensional image is produced by acquiring the image data in layers, the path of the scanning light beam ideally describing a meander on or in the object. To permit the acquisition of image data in layers, once a layer is scanned, the specimen stage or the objective is shifted, thereby bringing the next layer to be scanned into the focal plane of the objective.
An enhanced resolution in the direction of the optical axis is able to be achieved by a double objective arrangement (4Pi arrangement), as described in the European Patent No. EP 0 491 289 entitled “Double-Confocal Scanning Microscope.” The light coming from the illuminating system is split into two partial beams which, counterpropagating through two mirror-symmetrically positioned objectives, simultaneously illuminate the specimen. The two objectives are positioned on different sides of their common object plane. This interferometric illumination produces an interference pattern in the object point that, in the context of constructive interference, has a primary maximum and a plurality of secondary maxima. This is referred to as “type A” 4Pi microscopy when there is only interference of the excitation light, “type B” when there is interference of the detected light, and “type C” in the case of simultaneous interference of the excitation light and the detected light. Because of the interferometric illumination, this double-confocal scanning microscope is able to obtain an axial resolution that is enhanced over that of conventional scanning microscopes.
An optical arrangement for illuminating objects, in particular fluorescing objects in a double-confocal scanning microscope, is known from the German Patent Application No. DE 100 46 410 A1. The scanning microscope has a component which unites the two detection beam paths, the light coming from the object being combinable in an at least substantially overlapping fashion in one propagation direction at the component which unites the detection beam path, and means being provided for influencing the phase of the light coming from the object which are positioned at least in one partial beam path of the detection beam path. The uniting component is positioned to permit a substantially lossless combining of the detected light by properly adjusting the phase of the detected light within at least one of the two detection beam paths.
The inherent disadvantage of the known double-confocal scanning microscopes (4Pi microscopes) is that unused excitation light, as well as detected light, are coupled out of the interferometric beam path undesirably and are lost, unused.